This competitive revision is being submitted for Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availablity of Recovery Act Funds for Competitive Revision Applications. Background. The ability of short interfering RNAs (siRNAs) to recognize mRNA is widely appreciated. siRNAs are entering Phase 1 trials, but their ultimate potential to impact human health is unclear. The overall goal of the parent proposal GM 77253 was to investigate the action of duplex RNAs that are complementary to gene promoters. GM 77253 had four Aims: i. Evaluate involvement of argonaute proteins in RNA-mediated gene activation ii. Characterize the mechanism of transcriptional silencing by agRNAs iii. Define rules for targeting gene promoters iv. Identify endogenous miRNAs that inhibit transcription Rationale for a significant expansion of research scope. Recent transcriptome studies have revealed many noncoding transcripts overlapping the 3'terminus of genes. The function of these transcripts is unknown and the potential for regulation downstream from the 3'-UTR has attracted little attention. While pursuing Aim ii, we observed that promoter-targeted RNAs were binding to a noncoding transcript that overlaps the 5'terminus of our target gene progesterone receptor. We hypothesized that RNAs that target sequences past the 3'-terminus of genes might also be able to bind noncoding transcripts and modulate gene expression. We characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3'end of the gene. Small RNAs complementary to a noncoding transcript inhibit PR transcription in T47D cells (a cell line with high PR expression) and activate PR transcription in MCF7 cells (a cell line with low PR expression). The RNAs recruit argonaute to the 3'- noncoding transcript, alter levels of RNA polymerase, and modulate transcription of PR pre-mRNA and mature mRNA. RNAs complementary to sequences beyond the 3'mRNA terminus of BRCA1 also inhibit gene expression. Our results extend the potential for RNA-mediated gene regulation to regions downstream from the 3'end of genes. We propose to characterize the mechanism of action of 3'-targeted RNAs to understand how they can modulate transcription even though they are not complementary to mRNA and target sequences that are far distant from gene promoters. PUBLIC HEALTH RELEVANCE: Regulation of gene expression is fundamental to biological processes. Our research investigates regions of chromosomal DNA that lie beyond the termini of genes. These regions have received little attention and we will investigate whether gene regulation can extend to these regions.